PROJECT SUMMARY Inflammatory and immune diseases arise due to aggressive inflammation or defects in the regulation of inflammatory pathways and result in tissue damage and dysfunction. Drug treatment regimens used to treat these diseases are often ineffective due to the complex pathogenesis and the inability to regulate the numerous signaling pathways involved in inflammatory responses and tissue healing. Mesenchymal stem cells (MSCs) offer a potent cell therapy for the treatment of inflammatory and immune disorders due to their ability to regulate complex inflammatory responses, largely through paracrine mechanisms by secreting various cytokines. MSC secreted paracrine factors not only suppress pro-inflammatory responses but also promote endogenous anti-inflammatory cell phenotypes and therefore, have the potential to regulate the multiple signaling pathways and cell types that contribute to the complex pathogenesis of inflammatory and immune diseases. However, lack of a robust therapeutic response to MSCs is observed in disease models, in part due to inconsistent cell numbers at sites of inflammation. Transplantation of MSC spheroids, which increases retention of cells at transplant sites, may offer a means of improving MSC-based therapies for inflammatory diseases. Additionally, MSC immunomodulation can be influenced by the cellular microenvironment, such as the presence of inflammatory cytokines, therefore manipulating physical and chemical elements of the MSC microenvironment may serve as a novel and simple means of enhancing MSC secretion of immunomodulatory paracrine factors. Therefore, the primary objective of this grant proposal is to regulate human MSC (hMSC) immunomodulation and paracrine secretion through the engineering of transplantable 3D stem cell microenvironments to enhance the efficacy of hMSC-based therapies for the treatment of inflammatory diseases. The central hypothesis of this proposal is that presentation of cytokines within 3D hMSC aggregates will enhance paracrine secretion to suppress inflammatory responses and promote endogenous anti- inflammatory phenotypes in an acute animal model of inflammatory bowel disease (IBD). This proposal is significant because it examines the ability to engineer the physical and biochemical elements of 3D MSC microenvironments in order to direct MSC paracrine factor secretion and enhance immunomodulatory capability in vivo. This proposal is innovative because these results will establish a novel approach for engineering the MSC microenvironment to produce a therapeutically relevant cell population in vivo that can be used to combat a variety of different inflammatory and immune diseases.